Structural basis of GPCR homodimer formation Accumulating evidence suggests that GPCRs are able to form dimers and/or higher-order oligomeric complexes. Class A receptors represent by far the largest subfamily of GPCRs, consisting of 670 members in humans. Several studies suggest that class A receptor homodimers/oligomers are endowed with functional properties that differ from the monomeric receptor species. A proper understanding of how class A GPCRs function at the molecular level requires the identification of the structural elements governing the dimerization/oligomerization of this class of receptors. Many studies suggest that residues located on the 'outer'(lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A GPCR dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer (BRET) saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5, 7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the BRET data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Since all class A GPCRs share a high degree of structural homology, these findings should be of broad general interest. Use of yeast expression technology to identify novel M3R-interacting proteins GPCRs do not function in isolation but are part of an intricate network of receptor-protein interactions that are responsible for modulating various aspects of GPCR pharmacology, function, localization, and stability. We recently initiated a project to identify proteins that can interact with and modulate the function of the M3R. Such proteins may represent attractive novel targets for the treatment of various pathophysiological conditions including type 2 diabetes and colon cancer. Specifically, we employed the split ubiquitin membrane-based yeast two-hybrid system (Thaminy et al., Meth Mol Biol 261, 297-312, 2004) to screen for M3R-interacting proteins. The main advantage of this system, as compared to traditional yeast two-hybrid screening approaches, is that the bait protein (the full-length M3R or any other GPCR) is localized to the plasma membrane (or other cellular membranes) and proteins that are able to interact with the bait GPCR are identified by the use of simple growth and colorimetric assays. Using this novel experimental approach (split ubiquitin membrane-based yeast two-hybrid system), we identified several novel M3R-interacting protein, including the putative membrane protein Tmem147. The amino acid sequence of Tmem147 is highly conserved among mammals but its physiological roles are unknown at present. We initially demonstrated that Tmem147 associates with M3Rs in co-transfected mammalian cells. Microscopic studies showed that the Tmem147/M3R interaction occurred in the ER, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we performed studies with H508 human colon cancer cells which endogenously express M3Rs and Tmem147. Treatment of H508 cells with a muscarinic agonist (carbachol) promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Strikingly, siRNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation, probably due to an increase in the number of cell surface M3Rs. The observation that Tmem147 represents a potent negative regulator of M3R function may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.